Ciliogenesis in cryopreserved mammalian tracheal epithelial cells cultured at the air–liquid interface

Abstract

To determine air–liquid interface (ALI) culture derived from cryopreserved mammalian tracheal ciliated cells is a viable ciliated cell model for the investigations of regulatory mechanisms of ciliary beat frequency (CBF), two studies were performed using ovine and porcine tracheae obtained from local slaughterhouses. The protease-digested tracheal ciliated cells were harvested and cultured at the ALI using collagen-coated, porous membrane inserts. In study 1, the ALI culturing protocols were established using non-cryopreserved ovine tracheal ciliated cells. Ciliogenesis was documented with immuno-histology and electron micrographs. Vigorous beating cilia were video-recorded. CBF was measured by laser light scattering. The functional integrity of the autonomic receptors of the ciliated cells was confirmed with the stimulatory responses of CBF using luminal methacholine and basolateral terbutaline. In study 2, porcine tracheal ciliated cells stored in liquid nitrogen for a minimum of 4 weeks were used. The cryopreserved cells were thawed and cultured using the ALI protocol established in study 1. After two months, cilia outgrowths were confirmed using video microscopy and scanning electron micrograph (SEM). The trans-epithelial resistances were 28.5kΩ (n=4). Luminal applications of 1μM and 10μM methacholine stimulated CBF from a baseline of 7.4±0.2Hz to 8.4±0.8Hz and 7.7±0.4Hz, respectively (n=5). Basolateral applications of 1μM and 10μM terbutaline stimulated CBF from a baseline of 7.5±0.3Hz to 8.2±0.4Hz and 8.0±0.4Hz, respectively (n=5). These data demonstrated that a ciliated cell bank can be established using cryopreserved ciliated cells for pulmonary drug discovery and toxicological screening.